Ehd1gene promoting plant flowering, and utlization thereof

ABSTRACT

A detailed linkage analysis of the Ehd1 region was performed with a large segregating population, essential for map-based cloning. As a result, the Ehd1 gene promoting rice heading (flowering) was successfully isolated. Rice heading time was also found to be altered upon introduction of this gene. Based on these facts, the newly isolated and identified Ehd1 gene is expected to be useful in promoting plant flowering.

TECHNICAL FIELD

The present invention relates to Ehd1 genes that promote plantflowering, and utilization thereof.

BACKGROUND ART

Rice heading time (flowering time) is mainly determined by photoperiodsensitivity that depends on day length and other factors (basalvegetative growth or temperature sensitivity). Genetic analysis ofheading time has been performed for some time, and to date, headingtime-associated genes such as Se1 locus (chromosome 6), E1 locus(chromosome 7), E2 locus (unknown), E3 locus (chromosome 3), or Ef1locus (chromosome 10) (Kinoshita, Rice Genetics Newsletter 15: 13-74,1998; Nishida et al., Ann. Bot. 88: 527-536, 2001) have been discoveredusing mutations and variations inherent to rice cultivars. Recently, theuse of DNA markers in rice genetic analyses has advanced the geneticanalysis of characteristics such as heading time that exhibits complexinheritance (mapping of quantitative trait loci (QTLs)) (Yano et al.,Plant Physiol. 127: 1425-1429, 2001). Genes associated with ricephotoperiod sensitivity have been isolated based on this work (Yano etal., Plant Cell 12: 2473-2484, 2000; Takahashi et al., PNAS 98:7922-7927, 2001; Kojima et al., Plant Cell Physiol. 43: 1096-1105, 2002;Yano, Curr. Opin. Plant Biol. 4: 130-135, 2001). Attempts to use theseisolated and identified rice-heading-time-associated genes to elucidategenetic control mechanisms are also progressing (Izawa et al., Gene Dev.16: 2006-2020, 2002; Hayama et al., Nature 422: 719-722, 2003). On theother hand, many cases of isolation and identification of genesassociated with plant flowering have been reported in Arabidopsisthaliana (Simpson, G. G. and Dean, C., Science 296: 285-289 (2002);Mouradov, A. et al., Plant Cell (Suppl.) 14: S111-130 (2002)).Furthermore, methods for controlling flowering time in Arabidopsisthaliana (a plant) using these genes have been proposed (PublishedJapanese Translation of International Publication Nos.: 2002-511270;2002-532069; 2002-537768; 2000-512845; Hei 11-512289; Hei 11-506001; andHei 10-508481). At the same time, a method for using rice genes to alterthe flowering time of Arabidopsis thaliana (a plant) has also beensuggested (Published Japanese Translation of International PublicationNo. 2002-335970). However, a great number of the genes associated withrice heading time remain to be isolated and identified.

DISCLOSURE OF THE INVENTION

The present invention was made under these circumstances. An objectiveof the present invention is to provide novel genes that regulate plantflowering. Another objective of this invention is to modify plantflowering time using these genes.

The Ehd1 locus is a quantitative trait locus (QTL) associated withheading time. It was detected using the progeny of a cross betweenjaponica rice cultivar “Taichung 65” and West African region ricecultivar “O. glaberrima Steud.” (IRGC 104038). The Ehd1 locus has beenproven to be located on the long arm of chromosome 10. Furthermore,analysis using a nearly isogenic line of the Ehd1 region (the IRGC104038 allele), which comprises a “Taichung 65” genetic background,proved that the Ehd1 locus is associated with heading promotion. Geneticstudies hitherto have also proved that the Ehd1 locus was mapped as asingle Mendelian locus (formally called Ef(t)) in the interval betweenRFLP markers C234 and G37, and co-segregated with C1369. However,isolating and identifying genes using map-based cloning was difficult atthe level of resolution of this linkage analysis.

The present inventors carried out a detailed linkage analysis of theEhd1 region with a large segregating population essential for map-basedcloning. A generation of progenies derived from backcrossing Taichung 65and IRGC 104038 was used as the segregating population for linkageanalysis. From these backcrossed progenies, those whose Ehd1 region washeterologous, and whose other genomic regions were mostly substitutedwith the Taichung 65 type genome were selected. From the 2500 progenyplants (F2 generation) produced by these selected plants byself-fertilization, plant having a chromosome with a recombination nearthe Ehd1 locus were selected using CAPS markers C1286 and G37, whichflank the Ehd1. Genotype of Ehd1 locus was determined by a progeny testof the F3 generation. As a result of linkage analysis, it wasdemonstrated that the Ehd1 was mapped between RFLP markers C814A andC234, and identified markers and eight and two recombinant individuals.

Since the nucleotide sequences of the RFLP markers flanking the Ehd1gene were found to be included in the published genomic nucleotidesequence information, the nucleotide sequence of the Ehd1 candidategenomic region was obtained from the published nucleotide sequence data.Using information on the nucleotide sequence of the candidate genomicregion, novel CAPS markers were created to narrow down the candidategene region. As a result, the Ehd1 candidate region was proved to beabout 16 kb, flanked by CAPS markers 26-28 and 12-14. Gene predictionsand similarity searches were carried out against the nucleotide sequenceof this candidate region proved the presence of three types of predictedgenes. One type showed similarity to the two-component responseregulator (ARR) gene of Arabidopsis. The other two types of predictedgenes were highly similar to rice EST, but shared no similarity to knowngenes with established functions. However, since none of these predictedgenes could be excluded as Ehd1 candidates, these three types ofpredicted genes were transformed to verify function as Ehd1 candidates.

For transformation, a BAC library was constructed from genomic DNAsderived from the indica rice cultivar Kasalath assumed to comprise afunctional Ehd1 allele. A BAC clone KBM128G10 comprising the Ehd1candidate gene was selected for use from the library. From the BAC cloneKBM128G10, an 11.5 kb BamHI fragment comprising the ARR-like candidategene and one of the predicted genes having a high similarity to the riceEST, and a 7.6 kb KpnI fragment comprising two predicted genes otherthan the ARR-like candidate gene, were excised. Each of these fragmentswas incorporated into a Ti-plasmid vector pPZP2H-lac, and introducedinto Taichung 65 via Agrobacterium. Regenerated plants were immediatelytransferred into growth chambers and cultivated under short-dayconditions to measure the number of days until their heading. Almost allof the transgenic plants (T₀) that were introduced with the 11.5 kbBamHI fragment headed earlier than those with the vector alone. On theother hand, the number of days to heading in plants introduced with the7.6 kb KpnI fragment was about the same as for plants with the vectoralone. Furthermore, transcription of the ARR-like Kasalath-derivedcandidate gene was observed in almost all of the plants introduced withthe 11.5 kb BamHI fragment. From these results the Ehd1 candidates couldthus be narrowed down to the ARR-like candidate gene comprised in the11.5 kb BamHI fragment.

Furthermore, to confirm that heading promotion was due to the transgene,plants with lower copy numbers of the gene were selected from thosetransgenic plants (T₀) with earlier heading. The inbred progeniesthereof were cultivated under short-day conditions to investigate thenumber of days until heading. Each of the three types of progenypopulations were respectively divided into early-maturing plants andlate-maturing plants. All the early-maturing plants retained thetransformed Kasalath-derived ARR-like candidate gene, and the number ofdays to their heading was essentially the same as for the nearlyisogenic line, T65 (Ehd1), in which the genetic background of Taichung65 had been substituted with the Ehd1 gene of O. glaberrima. On theother hand, the number of days to Taichung 65 heading was the same asthat of late-maturing plants. These results proved that the ARR-likecandidate gene was the Ehd1 gene.

In rice cultivars O. glaberrima (IRGC 104038), Kasalath, Nipponbare, andTaichung 65, genomic nucleotide sequences of about 7.6 kb in their Ehd1regions were analyzed to compare the amino acid sequences of theirrespective predicted translational products. It was revealed that sevenamino acids were substituted between IRGC 104038 and Taichung 65, twobetween Kasalath and Taichung 65, and one between Nipponbare andTaichung 65. Of these, the substitution of the 219^(th) amino acidglycine to arginine was a unique mutation occurring only in Taichung 65.This glycine was highly conserved among the known ARR gene family. Thisamino acid mutation was thus assumed to be associated with the reducedEhd1 function of Taichung 65-derived plants.

Therefore, it is expected that the newly isolated and identified Ehd1gene can be utilized to promote plant flowering (heading), and that DNAswhich elicit a reduced function of the Ehd1 gene can be used to delayplant flowering.

Namely, the present invention relates to the Ehd1 gene, which promotesplant flowering (heading), and to utilization thereof. Morespecifically, the present invention provides the following:

-   -   [1] A DNA according to any one of the following (a) through (d),        wherein said DNA encodes a plant-derived protein comprising a        function of promoting plant flowering:    -   (a) a DNA encoding a protein comprising the amino acid sequence        of SEQ ID NO: 3, 6, or 9;    -   (b) a DNA comprising a coding region of a nucleotide sequence of        SEQ ID NO: 1, 2, 4, 5, 7, or 8;    -   (c) a DNA encoding a protein comprising an amino acid sequence        of SEQ ID NO: 3, 6, or 9, wherein one or more amino acids are        substituted, deleted, inserted, and/or added; and    -   (d) a DNA hybridizing under stringent conditions with a DNA        comprising the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7,        or 8;    -   [2] The DNA of [1], derived from rice;    -   [3] A DNA according to any one of the following (a) through (d):    -   (a) a DNA encoding an antisense RNA complementary to the        transcriptional product of the DNA of [1] or [2];    -   (b) a DNA encoding an RNA comprising ribosomal activity that        specifically cleaves the transcriptional product of the DNA of        [1] or [2];    -   (c) a DNA encoding an RNA that, due to an RNAi effect,        suppresses the expression of the DNA of [1] or [2] upon        expression in a plant cell; and    -   (d) a DNA encoding an RNA that, due to a co-suppression effect,        suppresses the expression of the DNA of [1] or [2] upon        expression in a plant cell;    -   [4] The DNA of [1] or [2], used to promote plant flowering;    -   [5] The DNA of [3], used to delay plant flowering;    -   [6] A vector comprising the DNA of any one of [1] through [5];    -   [7] A transformed plant cell retaining the DNA of any one of [1]        through [5] or the vector of [6];    -   [8] A transgenic plant comprising the transformed plant cell of        [7];    -   [9] A transgenic plant that is a progeny or a clone of the        transgenic plant of [8];    -   [10] A breeding material of the transgenic plant of [8] or [9];    -   [11] A method for producing the transgenic plant of [8],        comprising the steps of introducing the DNA of any one of [1]        through [5] or the vector of [6] into a plant cell, and        regenerating a plant from said plant cell;    -   [12] A method for promoting plant flowering, comprising        expressing the DNA of [1] or [2] in plant cells;    -   [13] A method for delaying plant flowering, comprising        suppressing the expression of the endogenous DNA of [1] or [2]        in plant cells;    -   [14] The method of [13], comprising introducing the DNA of [3]        into a plant; and    -   [15] The method of any one of [12] through [14], wherein the        plant is rice.

The present invention provides DNAs encoding plant-derived Ehd1 proteinscomprising the function of promoting plant flowering.

In this invention, the plants from which DNAs encoding the Ehd1 proteinare derived include, but are not limited to, rice, Arabidopsis thaliana,soybean, maize, barley, wheat and morning glory, for example.

Furthermore, there is no particular limitation as to the types of plantswhose flowering is promoted on transforming the above-described DNA. Forexample, such plants include useful crops and ornamental plants.Specifically, useful crops include monocotyledons such as rice, anddicotyledons such as soybean. Ornamental plants include flowering plantssuch as chrysanthemum, morning glory, poinsettia, and cosmos.

In the present invention, “flowering” usually means the blooming offlowers, but refers to heading in gramineous plants such as rice. Inthis invention, promotion of flowering refers to accelerating floweringtime, while delay of flowering refers to delaying flowering time.

Furthermore, the day-length (photoperiod) conditions under which theDNAs of this invention promoted the flowering of the above-describedplants are, for example, natural day-length conditions, long-dayconditions, short-day conditions, and so on. Short-day conditions arepreferred. In this invention, long-day conditions are conditions inwhich there are 14 or more daylight hours per day. In this example, thelight period was set at 15 hours, while the dark period was set at ninehours; however, long-day conditions are not limited to this example.Short-day conditions are those conditions in which there are 11 or fewerdaylight hours per day. In this example, the light period was set at tenhours and the dark period at 14 hours; however, short-day conditions arenot limited to this example.

Furthermore, in this invention, examples of DNAs encoding Ehd1 proteinsare those DNAs comprising the coding region of the nucleotide sequencesset forth in SEQ ID NO: 1, 2, 4, 5, 7, or 8, and DNAs encoding proteinscomprising the amino acid sequences set forth in SEQ ID NO: 3, 6, or 9.

The present invention also comprises DNAs encoding proteins which arestructurally analogous to an Ehd1 protein comprising an amino acidsequence set forth in SEQ ID NO: 3, 6, or 9, and comprising the functionof promoting plant flowering. Whether or not a DNA encodes a proteinthat comprises the function of promoting plant flowering can bedetermined by, for example, observing whether or not the flowering ofplants introduced with the DNA is promoted; or whether or not theflowering of plants is delayed when they are introduced with a DNA thatsuppresses the expression of the DNA. Examples of such DNAs includemutants, derivatives, alleles, variants, and homologues that encodeproteins comprising an amino acid sequence of SEQ ID NO: 3, 6, or 9, inwhich one or more of the amino acids are substituted, deleted, added,and/or inserted.

Examples of methods for preparing DNAs that encode proteins comprisingaltered amino acid sequences are well known to those skilled in the art,and include site-directed mutagenesis (Kramer, W. and Fritz, H.-J.,(1987) “Oligonucleotide-directed construction of mutagenesis via gappedduplex DNA.” Methods in Enzymology, 154: 350-367). A protein's aminoacid sequence may also mutate naturally due to a nucleotide sequencemutation. DNAs encoding proteins comprising an amino acid sequence of anEhd1 protein wherein one or more amino acids are substituted, deleted,added, and/or inserted are also included in the DNAs encoding Ehd1proteins of the present invention, so long as they encode a proteinfunctionally equivalent to a naturally occurring type of Ehd1 protein(SEQ ID NO.: 3, 6, or 9). In addition, nucleotide sequence mutationsthat do not give rise to changes in the amino acid sequence of theprotein (degenerate mutations) are also included in the DNAs encodingEhd1 proteins of the present invention.

DNAs encoding proteins functionally equivalent to Ehd1 proteins, whichcomprise an amino acid sequence described in SEQ ID NO: 3, 6, or 9, canbe produced, for example, by methods well known to those skilled in theart, including hybridization techniques (Southern, E. M. (1975) Journalof Molecular Biology 98: 503.); and polymerase chain reaction (PCR)techniques (Saiki, R. K. et al. (1985) Science 230: 1350-1354; Saiki, R.K. et al. (1988) Science 239: 487-491). That is, it is routine for aperson skilled in the art to isolate DNAs with high homology to a DNAencoding Ehd1 protein from rice and other plants, using the genomicsequence of an Ehd1 region (SEQ ID NO: 1, 4, or 7), an Ehd1 cDNAsequence (SEQ ID NO: 2, 5, or 8) or parts thereof as a probe, andoligonucleotides hybridizing specifically to the genomic sequence of theEhd1 region and the Ehd1 cDNA sequence as a primer. Such DNAs encodingproteins functionally equivalent to an Ehd1 protein, obtainable byhybridization techniques or PCR techniques, are also included in theDNAs encoding Ehd1 proteins of this invention.

Hybridization reactions to isolate such DNAs are preferably conductedunder stringent conditions. Stringent hybridization conditions of thepresent invention include conditions such as 6 M urea, 0.4% SDS, and0.5×SSC, and those conditions yielding similar stringencies to these.DNAs with higher homology are expected to be isolated when hybridizationis performed under more stringent conditions, for example, 6 M urea,0.4% SDS, and 0.1×SSC. Herein, high homology means identity over theentire amino acid sequence of at least 50% or above, more preferably 70%or above, much more preferably 90% or above, and most preferably 95% orabove.

The degree of homology of one amino acid sequence or nucleotide sequenceto another can be determined using the BLAST algorithm by Karlin andAltschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl.Acad. Sci. USA 90: 5873-5877, 1993). Programs such as BLASTN and BLASTX,developed based on the BLAST algorithm (Altschul et al. (1990) J. Mol.Biol. 215: 403-410), are also being used. To analyze a nucleotidesequence according to BLASTN, parameters are set, for example, atscore=100 and word length=12. On the other hand, parameters used for theanalysis of amino acid sequences by BLASTX are, for example, score=50and word length=3. The default parameters for each program are used whenusing the BLAST and Gapped BLAST programs. Specific techniques for suchanalysis are known in the art (see http://www.ncbi.nlm.nih.gov.)

DNAs of the present invention include genomic DNAs, cDNAs, andchemically synthesized DNAs. Genomic DNAs and cDNAs can be preparedaccording to conventional methods known to those skilled in the art.More specifically, genomic DNAs can be prepared, for example, by (1)extracting genomic DNAs from rice cultivars that comprise a DNA encodingan Ehd1 protein; (2) constructing a genomic library (utilizing a vector,such as a plasmid, phage, cosmid, BAC, PAC, and so on); (3) expandingthe library; and (4) conducting colony hybridization or plaquehybridization using a probe prepared based on a DNA that encodes an Ehd1protein of the present invention (e.g. SEQ ID NO.: 1, 2, 4, 5, 7, or 8).Alternatively, genomic DNAs can be prepared by PCR, using primersspecific to a DNA encoding an Ehd1 protein of the present invention(e.g. SEQ ID NO.: 1, 2, 4, 5, 7, or 8). cDNAs can be prepared, forexample, by (1) synthesizing cDNAs based on mRNAs extracted from ricecultivars that comprise a DNA encoding an Ehd1 protein; (2) preparing acDNA library by inserting the synthesized cDNA into vectors, such asλZAP; (3) expanding the cDNA library; and (4) conducting colonyhybridization or plaque hybridization as described above. Alternatively,cDNAs can be also prepared by PCR.

DNAs encoding an Ehd1 protein of the present invention can be used topromote plant flowering, for example. To prepare transgenic plants withpromoted flowering, the above-described DNAs are inserted intoappropriate vectors and introduced into plant cells using a methoddescribed below. Transgenic plants are regenerated from the transformedplant cells thus obtained. The present invention provides such methodsfor promoting plant flowering.

The present invention also provides methods for delaying plantflowering. Transgenic plants with delayed flowering can be obtained, forexample, by inserting a DNA which suppresses the expression of an Ehd1protein-encoding DNA into an appropriate vector; transforming the DNAconstruct into a plant cell using a method described below; andregenerating a plant from the resulting transformed plant cell. The stepof suppressing the expression of Ehd1 protein-encoding DNAs includessuppressing the transcription of these DNAs, as well as theirtranslation into proteins. In addition, it includes not only completehalting of DNA expression but also its reduction. It also includes theloss of an in vivo function of the translated proteins in plant cells.

Antisense techniques are the most commonly used methods in the art tosuppress the expression of a specific endogenous gene in plants. Eckeret al. were the first to demonstrate the antisense effect of anantisense RNA introduced into plant cells by electroporation (J. R.Ecker and R. W. Davis (1986) Proc. Natl. Acad. Sci. USA 83: 5372). Afterthat, expression of antisense RNAs reportedly reduced target geneexpression in tobacco and petunias (A. R. van der Krol et al. (1988)Nature 333: 866). Antisense techniques have now been established as ameans for suppressing target gene expression in plants.

Multiple factors act in the suppression of target gene expression byantisense nucleic acids. These include: inhibition of transcriptioninitiation by triple strand formation; inhibition of transcription byhybrid formation at a site where the RNA polymerase has formed a localopen loop structure; transcription inhibition by hybrid formation withthe RNA being synthesized; inhibition of splicing by hybrid formation atan intron-exon junction; inhibition of splicing by hybrid formation at asite of spliceosome formation; inhibition of mRNA translocation from thenucleus to the cytoplasm by hybrid formation with mRNA; inhibition ofsplicing by hybrid formation at a capping site or poly A addition site;inhibition of translation initiation by hybrid formation at atranslation initiation factor binding site; inhibition of translation byhybrid formation at a ribosome binding site near the initiation codon;inhibition of peptide chain elongation by hybrid formation in atranslated region or at an mRNA polysome binding site; and inhibition ofgene expression by hybrid formation at a site of interaction betweennucleic acids and proteins. These antisense nucleic acids suppresstarget gene expression by inhibiting various processes such astranscription, splicing, or translation (Hirashima and Inoue, “ShinSeikagaku Jikken Koza (New Biochemistry Experimentation Lectures) 2,Kakusan (Nucleic Acids) IV, Idenshi No Fukusei To Hatsugen (Replicationand Expression of Genes),” Nihon Seikagakukai Hen (The JapaneseBiochemical Society), Tokyo Kagaku Dozin, pp. 319-347, (1993)).

The antisense sequences of the present invention can suppress targetgene expression by any of the above mechanisms. In one embodiment, anantisense sequence designed to be complementary to an untranslatedregion near the 5′ end of the mRNA of a gene is thought to effectivelyinhibit translation of that gene. Sequences complementary to codingregions or to an untranslated region on the 3′ side can also be used.Thus, the antisense DNAs used in the present invention include both DNAscomprising antisense sequences against untranslated regions and againsttranslated regions of the gene. The antisense DNAs to be used areconjugated downstream of an appropriate promoter, and are preferablyconjugated to sequences containing the transcription termination signalon the 3′ side. DNAs thus prepared can be transformed into a desiredplant by known methods. The sequences of the antisense DNAs arepreferably sequences complementary to an endogenous gene of the plant tobe transformed, or a part thereof, but need not be perfectlycomplementary so long as they can effectively suppress the gene'sexpression. The transcribed RNAs are preferably at least 90%, and morepreferably at least 95% complementary to the transcribed product of thetarget gene. In order to effectively suppress the expression of a targetgene by means of an antisense sequence, antisense DNAs should be atleast 15 nucleotides long, more preferably at least 100 nucleotideslong, and still more preferably at least 500 nucleotides long. However,the antisense DNAs to be used are generally shorter than 5 kb, andpreferably shorter than 2.5 kb.

DNAs encoding ribozymes can also be used to suppress the expression ofendogenous genes. A ribozyme is an RNA molecule comprising catalyticactivity. There are many ribozymes comprising various activities, andamong them, research focusing on ribozymes as RNA-cleaving enzymes hasenabled the design of ribozymes that cleave RNAs site-specifically.While some ribozymes of the group I intron type or the M1 RNA containedin RNaseP consist of 400 nucleotides or more, others belonging to thehammerhead-type or the hairpin-type comprise an activity domain of about40 nucleotides (Makoto Koizumi and Eiko Ohtsuka (1990) TanpakushitsuKakusan Kohso (Nucleic acid, Protein, and Enzyme) 35: 2191).

The self-cleavage domain of a hammerhead-type ribozyme cleaves at the 3′side of C15 of the G13U14C15 sequence, and formation of a nucleotidepair between U14 and A9 at the ninth position is considered to beimportant for this ribozyme activity. It has been shown that cleavagemay also occur when the 15th nucleotide is A15 or U15 instead of C15 (M.Koizumi et al. (1988) FEBS Lett. 228: 225). If a ribozyme is designed tocomprise a substrate-binding site complementary to the RNA sequencesadjacent to the target site, one can create a restriction-enzyme-likeRNA-cleaving ribozyme which recognizes the UC, UU, or UA sequence withina target RNA (M. Koizumi et al. (1988) FEBS Lett. 239: 285; M. Koizumiet al. (1989) Nucleic Acids Res. 17: 7059). For example, in DNAs thatencode Edh1 proteins (SEQ ID NO.: 2, 5, or 8), there are a number ofsites that can be used as ribozyme targets.

Hairpin-type ribozymes are also useful in the present invention. Theseribozymes can be found, for example, in the minus strand of satelliteRNA in tobacco ringspot virus (J. M. Buzayan (1986) Nature 323: 349).Ribozymes that cleave RNAs target-specifically have also been shown tobe produced from hairpin-type ribozymes (Y. Kikuchi and N. Sasaki (1992)Nucleic Acids Res. 19: 6751; Yo Kikuchi (1992) Kagaku To Seibutsu(Chemistry and Biology) 30: 112).

Transcription is enabled in plant cells by fusing a ribozyme, designedto cleave a target, with a promoter such as the cauliflower mosaic virus³⁵S promoter, and with a transcription termination sequence. If extrasequences have been added to the 5′ end or the 3′ end of the transcribedRNA, ribozyme activity can be lost. In such cases, one can place anadditional trimming ribozyme, which functions in cis, on the 5′ or the3′ side of the ribozyme portion, in order to precisely cut the ribozymeportion from the transcribed RNA containing the ribozyme (K. Taira etal. (1990) Protein Eng. 3: 733; A. M. Dzaianott and J. J. Bujarski(1989) Proc. Natl. Acad. Sci. USA 86: 4823; C. A. Grosshands and R. T.Cech (1991) Nucleic Acids Res. 19: 3875; K. Taira et al. (1991) NucleicAcid Res. 19: 5125). Even greater effects can be achieved by arrangingthese structural units in tandem, enabling multiple sites within atarget gene to be cleaved (N. Yuyama et al., Biochem. Biophys. Res.Commun. 186: 1271 (1992)). Thus, using these ribozymes, thetranscription products of a target gene of the present invention can bespecifically cleaved, thereby suppressing expression of the gene.

Endogenous gene expression can also be suppressed by RNA interference(RNAi), using double-stranded RNAs that comprise a sequence identical orsimilar to a target gene. RNAi refers to the phenomenon in which adouble-stranded RNA comprising a sequence identical or similar to atarget gene sequence is introduced into cells, thereby suppressingexpression of both the exogenous gene introduced and the targetendogenous gene. The details of the RNAi mechanism are unclear, but itis thought that an introduced double-stranded RNA is first degraded intosmall pieces, which somehow serve as a target gene indicator, resultingin degradation of the target gene. RNAi is known to be effective inplants as well (Chuang C F, Meyerowitz E M, Proc Natl Acad Sci USA 97:4985, 2000). For example, in order to use RNAi to suppress theexpression of DNAs that encode the Ehd1 protein in plants, Ehd1protein-encoding DNAs (SEQ ID: 2, 5, or 8), or double-stranded RNAscomprising a sequence similar to these DNAs, can be introduced into theplants in question. Plants whose flowering is delayed compared to awild-type plant can then be selected from the resulting plants. Genesused for RNAi need not be completely identical to a target gene;however, they should comprise sequence identity of at least 70% orabove, preferably 80% or above, more preferably 90% or above, and mostpreferably 95% or above. Sequence identity can be determined by anabove-described method.

Suppression of endogenous gene expression can be achieved byco-suppression, through transformation with a DNA comprising a sequenceidentical or similar to a target gene sequence. “Co-suppression” refersto the phenomenon wherein transformation is used to introduce plantswith a gene comprising a sequence identical or similar to a targetendogenous gene sequence, thereby suppressing expression of both theexogenous gene introduced and the target endogenous gene. Although thedetails of the co-suppression mechanism are unclear, at least a part isthought to overlap with the RNAi mechanism. Co-suppression is alsoobserved in plants (Smyth DR, Curr. Biol. 7: R793, 1997; Martienssen RCurr. Biol. 6: 810, 1996). For example, if one wishes to obtain a plantin which a DNA encoding an Ehd1 protein is co-suppressed, the plant inquestion can be transformed with a vector DNA designed to express theDNA encoding the Ehd1 protein, or a DNA comprising a similar sequence.Plants whose flowering is delayed compared to a wild-type plant are thenselected from the resultant plants. Genes for use in co-suppression donot need to be completely identical to a target gene, but shouldcomprise sequence identity of at least 70% or above, preferably 80% orabove, more preferably 90% or above, and most preferably 95% or above.Sequence identity may be determined by an above-described method.

The present invention provides methods for producing transgenic plants,wherein said methods comprise the steps of introducing a DNA of thepresent invention into plant cells, and regenerating plants from thesecells.

In the present invention, cells can be derived from any plant, withoutlimitation. Vectors used for the transformation of plant cells are notlimited as long as they can express the inserted gene in the plantcells. Vectors that can be used include, for example, vectors comprisingpromoters (e.g., cauliflower mosaic virus 35S promoter) for constitutivegene expression in plant cells, and vectors comprising promoters thatare inducibly activated by external stimuli. The term “plant cell” asused herein includes various forms of plant cells, such as culture cellsuspensions, protoplasts, leaf sections, and calluses.

A vector can be introduced into plant cells by known methods, such as byusing polyethylene glycol, electroporation, Agrobacterium-mediatedtransfer, and particle bombardment. Agrobacterium (for example, EHA101)mediated transfer can be carried out by, for example, the ultraspeedtransformation of monocotyledon (Japanese Patent No. 3141084). Particlebombardment can be carried out by, for example, using equipmentavailable from Bio-Rad. Plants can be regenerated from transformed plantcells by known methods and according to the type of the plant cell (seeToki et al., (1995) Plant Physiol. 100:1503-1507).

For example, methods for producing a transformed rice plant include: (1)introducing genes into protoplasts using polyethylene glycol, thenregenerating the plant (suitable for indica rice cultivars) (Datta, S.K. (1995) in “Gene Transfer To Plants”, Potrykus I and Spangenberg Eds.,pp 66-74); (2) introducing genes into protoplasts using electric pulses,then regenerating the plant (suitable for japonica rice cultivars) (Tokiet al. (1992) Plant Physiol. 100: 1503-1507); (3) introducing genesdirectly into cells by particle bombardment, then regenerating the plant(Christou et al. (1991) Bio/Technology, 9: 957-962); (4) introducinggenes using Agrobacterium, then regenerating the plant (Hiei et al.(1994) Plant J. 6: 271-282); and so on. These methods are alreadyestablished in the art and are widely used in the technical field of thepresent invention. Such methods can be suitably used in the presentinvention.

Once a transgenic plant is obtained, in which a DNA of the presentinvention has been introduced into its genome, progenies can be derivedfrom that plant by sexual or vegetative propagation. Alternatively,plants can be mass-produced from breeding materials obtained from theplant (for example, from seeds, fruits, ears, tubers, tubercles, tubs,calluses, protoplasts, etc.), as well as from progenies or clonesthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the Ehd1 locus on chromosomes.

FIG. 2 is a bar graph showing the number of days until heading of nearlyisogenic rice line T65(Ehd1) of the O. glaberrima Ehd1 gene, and itsrecurrent parent line (Taichung 65) under short-day, long-day, andnatural day-length conditions. Under short-day and long-day conditions,plants were cultivated without transplantation in a Kyushu Universityoutdoor day-length regulator. The results for natural day-lengthconditions are those of early season cultivation at Kyushu University(Fukuoka City) (sowed on May 2 and transplanted on June 14).

FIG. 3 represents a diagram showing a high-resolution linkage map andphysical map of the Ehd1 region:

A: a linkage map prepared using a segregating population of 2500individual plants and RFLP markers. The labels above the line representRFLP markers, and the numerals under the line indicate the number ofrecombinations detected in the intervals between respective markers;

-   -   B: a detailed linkage map using CAPS markers and prepared based        on nucleotide sequence information; and    -   C: a putative gene in the candidate genomic region, and genomic        DNA fragments used in the transformation.

FIG. 4 depicts bar graphs showing the frequency distribution of thenumber of days to heading of a transformed plant (T0) produced byintroducing either the 11.5 kb BamHI fragment or 7.6 kb KpnI fragment ofKasalath into Taichung 65, and cultivating under short-day conditions(ten hours of daylight).

FIG. 5 is a photograph showing the presence or absence of KasalathARR-like gene expression in the individual transformed plants. Total RNAwas extracted from each plant, reverse transcribed, PCR-amplified, anddigested with DdeI. The sizes of products corresponding to the Kasalathand Taichung 65-derived mRNAs are shown with “K” and “T” respectively.

FIG. 6 is a diagram showing the Ehd1 gene structure and nucleotidesequence polymorphism.

FIG. 7 shows a comparison of Ehd1 protein amino acid sequences. Thearrow indicates the position of the mutated amino acid found only inTaichung 65. The amino acid sequences of Taichung 65, Nipponbare,Kasalath, and IRGC 104038 are set forth in SEQ ID NOs. 12, 9, 6, and 3,respectively.

FIG. 8 represents a photograph and diagram showing changes in the amountof Ehd1 mRNA accumulated over a day. Four weeks after sowing, total RNAwas extracted from the leaves of Taichung 65 (T) and nearly isogenicline T65(Ehd1) (N) of the Ehd1 gene of O. glaberrima Steud. (IRGC104038), and RT-PCR analysis was performed over 30 PCR cycles.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention is more specifically described withreference to Examples; however, it is not to be construed as beinglimited thereto.

EXAMPLE 1 The Heading-Promoting Action of the Ehd1 Gene Under Short-DayConditions

The Ehd1 locus is a QTL associated with heading time and detected usingthe progeny of a cross between japonica rice cultivar “Taichung 65” andWest African region rice cultivar O. glaberrima Steud. (IRGC 104038).The Ehd1 locus has been proved to be located on the long arm ofchromosome 10 (Doi et al., Breeding Science 49: 395-399, 1999) (FIG. 1).The Ehd1 gene of O. glaberrima (IRGC 104038) has been shown to comprisea heading-promoting action, and acts dominantly over the allele ofTaichung 65. In this Example, both a nearly isogenic rice line T65(Ehd1), in which the genetic background of Taichung 65 had beensubstituted with the Ehd1 gene of O. glaberrima, and Taichung 65 werecultivated under different day-length conditions to investigate thenumber of days until their heading. T65(Ehd1) headed seven days earlierthan Taichung 65 under natural day-length conditions, 14 days earlierunder long-day conditions, and 33 days earlier under short-dayconditions (FIG. 2). These results demonstrated that the Ehd1 genecomprised the function of promoting heading, and that itsheading-promoting action becomes more prominent under short-dayconditions.

EXAMPLE 2 High-Resolution Linkage Analysis

Genetic analysis performed hitherto has proven that the Ehd1 locus ispositioned as a single gene locus (old name: Ef(t)) between RFLP markersC234 and G37, and co-segregated with C1369 (Doi, Taguchi, and Yoshimura:Japanese Society of Breeding, 94th lecture, Japanese Journal of Breeding(Suppl.), p 104, 1998). However, at the resolution level of this linkageanalysis, it was difficult to isolate and identify genes using map-basedcloning. In this example, detailed linkage analysis of the Ehd1 regionwas performed with a large segregating population essential formap-based cloning. A generation of progenies derived from backcrossingTaichung 65 and IRGC 104038 was used as the segregating population forlinkage analysis. From these backcrossed progeny, plants were selectedwhose Ehd1 region was heterologous, and whose other genomic regions weremostly substituted with Taichung 65 type genome. These selected plantsunderwent self-fertilization, producing 2500 progeny plants (the F2population). Those plants that comprise a chromosome with arecombination near the Ehd1 locus were selected using Ehd1-flanking CAPS(Cleaved Amplified Polymorphic Sequence) markers C1286 (primers [SEQ IDNO: 13/5′-CCAATGAAGGGTAAGTATCG-3′] and [SEQ ID NO:14/5′-TGTGCTTAAGATACACGGTAGTTCA-3′], restriction enzyme NruI); and G37(primers [SEQ ID NO: 15/5′-CTGCAGCTTCCACCATGGCA-3′] and [SEQ ID NO:16/5′-CAAGGGTGCATTCATTGCACCTCCTCTAGCCATGGCCTAATGATGCA-3′], restrictionenzyme EcoT22I) The genotype of the Ehd1 locus was determined by aprogeny test of the F3 generation. That is, 48 inbred progeny plantsfrom the selected plants (F2) were cultivated in an experimental farm atKyushu University to determine their Ehd1 genotype based on variationsin the number of days until the heading of each line. The linkageanalysis results located the Ehd1 locus between RFLP markers C814A andC234, identifying eight and two recombinant individuals between Ehd1 andthese markers respectively (FIG. 3).

EXAMPLE 3 Identification of a Candidate Gene Region

The nucleotide sequences of the RFLP markers flanking the Ehd1 gene werefound to be comprised in published genomic nucleotide sequences, suchthat the nucleotide sequence of Ehd1 candidate genomic region wasobtained from published nucleotide sequence data (GenBank Accession No.AC027038). Using information on the nucleotide sequences of thiscandidate genomic region, novel CAPS markers were prepared for narrowingdown the candidate genomic region. Ehd1 was found to have tworecombinations with CAPS marker 26-28 (primers [SEQ ID NO:17/5′-ACGCTGCAACAAAGAGCAGA-3′] and [SEQ ID NO:18/5′-TTGTTGACGAAAGCCCATTG-3′], restriction enzyme MspI); and onerecombination with 12-14 (primers [SEQ ID NO:19/5′-GGAGATCATGCTCACGGATG-3′] and [SEQ ID NO:20/5′-CAAGCAAACACGGAGCGACT-3′], restriction enzyme BamHI). Furthermore,the Ehd1 gene was co-segregated with CAPS markers 13-15 (primers [SEQ IDNO: 21/5′-CCTTGCATCCGTCTTGATTG-3′] and [SEQ ID NO:22/5′-GGGCAAATTCCCTCCAGAGT-3′], restriction enzyme MspI); 19-21 (primers[SEQ ID NO: 23/5′-TTTGGATACGTACCCCTGCAT-3′] and [SEQ ID NO:24/5′-GCGCAATCGCATACACAATAA-3′ ], restriction enzyme MspI); and 23-25(primers [SEQ ID NO: 25/5′-GAGCCCGAGCCCATGTATAG-3′] and [SEQ ID NO:26/5′-TGGCTAAGATGGAGGGACGA-3′], restriction enzyme MboI). Therefore, theEhd1 candidate region was narrowed down to a region of about 16 kb,flanked by CAPS markers 26-28 and 12-14. Gene predictions and similaritysearches carried out against the nucleotide sequence of this candidateregion using GENSCAN (http://genes.mit.edu/GENSCAN.html) proved thepresence of three types of predicted genes. One type showed similarityto the two-component response regulator (ARR) gene of Arabidopsis. Theother two types showed high similarity to rice EST, but no resemblanceto known genes whose functions had been established. However, since noneof these predicted genes could be excluded as Ehd1 candidates,transformation of these three types of predicted genes was used toverify function as Ehd1 candidates.

EXAMPLE 4 Functional Verification of Ehd1 Candidate Gene

A genomic DNA fragment derived from the indica rice cultivar Kasalath,which is assumed to comprise a functional Ehd1 allele, was used for thetransformation. That is, a BAC library constructed from the genomic DNAof Kasalath (Baba et al., Bulletin of the NIAR 14: 41-49, 2000) wasscreened using a pair of primers 10-12 (SEQ ID NO:27/5′-ATTGGGCCAAACTGCAAGAT-3′ and SEQ ID NO:28/5′-ACGAGCCTAATGGGGGAGAT-3′) capable of amplifying the nucleotidesequence near Ehd1 to select BAC clone KBM128G10, which comprises theEhd1 candidate gene. An 11.5 kb BamHI fragment comprising the ARR-likecandidate gene and one of the predicted genes showing high similarity tothe rice EST, and a 7.6 kb KpnI fragment comprising the two predictedgenes other than the ARR-like candidate genes (FIG. 3), were excisedfrom the BAC clone KBM128G10. These fragments were each incorporatedinto the Ti-plasmid vector pPZP2H-lac (Fuse et al., Plant Biotechnology18: 219-222, 2001), and these transformed vectors were then introducedinto Taichung 65, mediated by Agrobacterium. Regenerated plants wereimmediately transferred to growth chambers under short-day conditions(ten hours of daylight) and cultivated to investigate the number of daysuntil heading. Almost all of the transgenic plants (TO) (18 plants)introduced with the 11.5 kb BamHI fragment headed earlier than theplants introduced with the vector alone (six plants) (FIG. 4). On theother hand, the number of days to heading in the six plants introducedwith the 7.6 kb KpnI fragment was about the same as for the five plantsintroduced with the vector alone (FIG. 4). The expression of theKasalath-derived ARR-like candidate gene in plants introduced with the11.5 kb BamHI fragment was also confirmed by RT-PCR using agene-specific marker (primers [SEQ ID NO: 29/5′-GAGATCAACGGCCACCGAAG-3′]and [SEQ ID NO: 30/5′-GTCGAGAGCGGTGGATGACA-3′], restriction enzymeDdeI). Transcription of the Kasalath-derived ARR-like candidate gene wasobserved in almost all of the plants (FIG. 5). Thus, the Ehd1 candidatecould be narrowed down to the ARR-like candidate gene contained in the11.5 kb BamHI fragment. Furthermore, to confirm that heading promotionwas due to the transgene, individual plants comprising low copy numbersof the gene were selected from those transformed plants (T0) withearlier heading. Their self-fertilized progenies were cultivated undershort-day conditions to investigate the number of days until heading.Each of the three types of progeny populations were divided intoearly-maturing plants (41-70 days to heading) and late-maturing plants(81 days or more to heading). All of the early-maturing plants retainedthe introduced Kasalath-derived ARR-like candidate gene, and headed atabout the same time as the nearly isogenic rice line T65(Ehd1). On theother hand, the number of days to heading of Taichung 65 was 81 days ormore, about the same as for late-maturing plants (Table 1). From theabove-described results, the ARR-like candidate gene was shown tocomprise heading-promotion function under short-day conditions, and tobe the Ehd1 gene. TABLE 1 Progeny Number of days to heading (days)population 41-45 46-50 51-55 56-60 61-65 66-70 71-75 76-80 >81 TotalPopulation 4 21  2 — — — — — 5* 32 1 Population —  1  7 — 7 4 — — 8* 272 Population —  2 17 — 1 — — — 5* 25 3 Taichung 65 — — — — — — — — 5  5T65(Ehd1) —  1  2 — — — — — — 3*The introduced gene was not retained.

EXAMPLE 5 Nucleotide Sequence Analysis of the Ehd1 Candidate Gene

The genomic nucleotide sequences of approximately 7.6 kb Ehd1 regionswere analyzed in rice cultivars O. glaberrima (IRGC 104038), Kasalath,Nipponbare, and Taichung 65 (TA repeats of 200 bp or more exist inTaichung 65; the exact number of these has not been determined).Compared to Kasalath, 60 or more nucleotide substitutions, insertions,and deletions were detected in this region in Nipponbare and Taichung65, and at 140 positions or more in IRGC 104038 (FIG. 6). Thefull-length cDNA of IRGC 104038 was also determined, and as a result ofcomparing the genomic nucleotide sequences, Ehd1 was predicted tocomprise six exons, with a full-length transcriptional region of 1316 bpand encoding a protein comprising 341 amino acids (FIG. 7). Comparisonof differences in nucleotide sequences within the predictedtranscriptional region with that of Kasalath detected foursingle-nucleotide substitutions and two-nucleotide insertions inNipponbare; five single-nucleotide substitutions and two-nucleotideinsertions in Taichung 65; and fourteen single-nucleotide substitutionsand four- and three-nucleotide deletions in IRGC 104038. On comparingthe amino acid sequences of the predicted translational products of IRGC104038, Kasalath, and Nipponbare, and Taichung 65, it was revealed thatseven amino acids are substituted between IRGC 104038 and Taichung 65,two amino acids between Kasalath and Taichung 65, and one amino acidbetween Nipponbare and Taichung 65 (FIG. 7). Of these, the substitutionof glycine at the 219^(th) amino acid (G in IRGC 104038, Kasalath, andNipponbare) to arginine (R in Taichung 65) was the only mutationoccurring in Taichung 65 alone (FIG. 7). This glycine had been highlyconserved among the known ARR gene families. These facts suggested thatthis amino acid mutation is associated with the reduced function of Ehd1derived from Taichung 65.

The genomic nucleotide sequences of the Ehd1 regions of O. glaberrima(IRGC 104038), Kasalath, Nipponbare, and Taichung 65 are set forth inSEQ ID NOs: 1, 4, 7, and 10 respectively; the nucleotide sequences ofcDNAs thereof are set out in SEQ ID NOs: 2, 5, 8, and 11 respectively;and the amino acid sequences of the proteins encoded by these DNAs areset out in SEQ ID NOs: 3, 6, 9, and 12, respectively.

EXAMPLE 6 Expression Analysis of the Ehd1 Candidate Gene

Variations of the Ehd1 gene expression level over a one-day period wereexamined for T65(Ehd1) in which the Ehd1 region of Taichung 65 had beensubstituted with a chromosomal fragment derived from IRGC 104038.T65(Ehd1) and Taichung 65 were sowed in experimental greenhouses (atTsukuba City, Ibaraki Prefecture) during the last ten days of December(sunrise: 6:50AM, sunset: 4:30PM; short-day conditions), cultivated forfour weeks, and then the leaves were collected from both rice linesevery three hours over 24 hours for analysis. Total RNA was extractedfrom these materials, and RT-PCR was performed for the Ehd1 gene using apair of primers (sense strand [SEQ ID NO: 31/5′-TGGATCACCGAGAGCTGTGG-3′]and an antisense strand [SEQ ID NO: 32/5′-ATTTCCTTGCATCCGTCTTG-3′]). Asa result, the transcriptional product of this gene was found toaccumulate in large amounts around dawn (3 and 6 o'clock), with atendency to decrease to undetectable levels after sunset till midnight(18 and 21 o'clock) (FIG. 8). Such circadian fluctuation in geneexpression level is a phenomenon often observed in genes associated withphotoperiod sensitivity, indicating some sort of association of thisEhd1 gene with photosignal transduction. From the above-describedresults, the candidate gene identified by map-based cloning was judgedto be the Ehd1 gene promoting rice heading.

INDUSTRIAL APPLICABILITY

In conventional rice breeding, flowering (heading) time has been alteredby (1) selection of early-maturing and late-maturing varieties bycrossing, (2) mutagenesis by radiation and chemicals, and so on.However, these procedures posed problems such as the requirement of longperiods of time for successful breeding, and the inability to controlthe degree or direction of variation. The present invention has enabledthe establishment of a novel method for modifying plant flowering timeby utilizing the isolated Ehd1 gene. Therefore, by transforming a sensestrand of Ehd1 gene into a plant cultivar in which the function of thisgene has been lost, for example, into Taichung 65 rice, rice heading(flowering) can be promoted under short-day conditions. At the sametime, by introducing the Ehd1 gene in the antisense direction into aplant cultivar in which the function of the Ehd1 gene is retained, forexample, into Nipponbare or Kasalath rice, expression of the endogenousEhd1 gene can be suppressed to delay heading (flowering). Since thisalteration can be expected to occur not only under short-day conditionsbut also under long-day or natural day-length conditions, the presentinvention is effective in controlling heading time (flowering time)under various cultivation conditions. Since the period of time requiredfor transformation is extremely short compared to that required for genetransfer by crossing, flowering time can be modified without alteringother characteristics. The use of the isolated Ehd1 gene to promoteflowering is expected to be useful in simple alteration of the floweringtime of plants such as rice, and to contribute to the breeding of plantsadapted to different locations.

1. A DNA according to any one of the following (a) through (d), whereinsaid DNA encodes a plant-derived protein comprising a function ofpromoting plant flowering: (a) a DNA encoding a protein comprising theamino acid sequence of SEQ ID NO: 3, 6, or 9; (b) a DNA comprising acoding region of a nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7, or8; (c) a DNA encoding a protein comprising an amino acid sequence of SEQID NO: 3, 6, or 9, wherein one or more amino acids are substituted,deleted, inserted, and/or added; and (d) a DNA hybridizing understringent conditions with a DNA comprising the nucleotide sequence ofSEQ ID NO: 1, 2, 4, 5, 7, or
 8. 2. The DNA of claim 1, derived fromrice.
 3. A DNA according to any one of the following (a) through (d):(a) a DNA encoding an antisense RNA complementary to the transcriptionalproduct of the DNA of claim 1 or 2; (b) a DNA encoding an RNA comprisingribozyme activity that specifically cleaves the transcriptional productof the DNA of claim 1 or 2; (c) a DNA encoding an RNA that, due to anRNAi effect, suppresses the expression of the DNA of claim 1 or 2 uponexpression in a plant cell; and (d) a DNA encoding an RNA that, due to aco-suppression effect, suppresses the expression of the DNA of claim 1or 2 upon expression in a plant cell.
 4. The DNA of claim 1 or 2, usedto promote plant flowering.
 5. The DNA of claim 3, used to delay plantflowering.
 6. A vector comprising the DNA of claim 1 or
 2. 7. Atransformed plant cell retaining the DNA of claim 1 or
 2. 8. Atransgenic plant comprising the transformed plant cell of claim
 7. 9. Atransgenic plant that is a progeny or a clone of the transgenic plant ofclaim
 8. 10. A breeding material of the transgenic plant of claim
 8. 11.A method for producing a transgenic plant, comprising the steps ofintroducing the DNA of claim 1 or 2 into a plant cell, and regeneratinga plant from said plant cell.
 12. A method for promoting plantflowering, comprising expressing the DNA of claim 1 or 2 in plant cells.13. A method for delaying plant flowering, comprising suppressing theexpression of the endogenous DNA of claim 1 or 2 in plant cells.
 14. Amethod for delaying plant flowering, comprising introducing the DNA ofclaim 3 into a plant.
 15. The method of claim 12, wherein the plant isrice.
 16. The method of claim 13, wherein the plant is rice.
 17. Themethod of claim 14, wherein the plant is rice.
 18. A transformed plantcell retaining the vector of claim
 6. 19. A transgenic plant comprisingthe transformed plant cell of claim
 18. 20. A transgenic plant that is aprogeny or a clone of the transgenic plant of claim
 19. 21. A breedingmaterial of the transgenic plant of claim
 19. 22. A breeding material ofthe transgenic plant of claim
 20. 23. A method for producing atransgenic plant, comprising the steps of introducing the vector ofclaim 6 into a plant cell, and regenerating a plant from said plantcell.
 24. A breeding material of the transgenic plant of claim
 9. 25. Atransformed plant cell retaining the DNA of claim
 3. 26. A transgenicplant comprising the transformed plant cell of claim
 25. 27. Atransgenic plant that is a progeny or a clone of the transgenic plant ofclaim
 26. 28. A breeding material of the transgenic plant of claim 26.29. A breeding material of the transgenic plant of claim
 27. 30. Avector comprising the DNA of claim
 3. 31. A transformed plant cellretaining the vector of claim
 30. 32. A transgenic plant comprising thetransformed plant cell of claim
 31. 33. A transgenic plant that is aprogeny or a clone of the transgenic plant of claim
 32. 34. A breedingmaterial of the transgenic plant of claim
 32. 35. A breeding material ofthe transgenic plant of claim
 33. 36. A method for producing atransgenic plant, comprising the steps of introducing the DNA of claim 3into a plant cell, and regenerating a plant from said plant cell.
 37. Amethod for producing a transgenic plant, comprising the steps ofintroducing the vector of claim 30 into a plant cell, and regenerating aplant from said plant cell.